The Na,K-ATPase is responsible for controlling cellular fluid and electrolyte balance in higher eukaryotes. It is a heterodimeric integral membrane protein consisting of a catalytic [unreadable]-subunit (~110 kDa) and a glycosylated 2-subunit (~55 kDa). During some physiological states, this single enzyme is responsible for utilizing nearly 40% of the cells energy. In kidney and intestinal epithelial cells the Na,K-ATPase is strictly delivered to the basolateral membrane providing directional uptake of Na+ and other solutes (e.g. glucose, amino acids). Recently, several laboratories have reported that in addition to solute transport, the Na,K-ATPase is a cell-surface receptor for endogenous cardiac glycoside-mediated Src signaling. Some of these alternative physiological roles of the Na,K-ATPase mandate that the enzyme be delivered to specific sub-plasma membrane pools, which raises as yet unaddressed questions pertaining to Na,K- ATPase maturation and trafficking. The planned experiments in this proposal will focus on the maturation and assembly of Na,K-ATPase, focusing on the role that oligomerization plays in membrane delivery. Our preliminary data has led to the overall hypothesis: Na,K-ATPase oligomerization is critical for proper membrane delivery. We will test this hypothesis in the proposed studies. The specific experiments outlined in this proposal will exploit insect cell expression, devoid of endogenous Na,K-ATPase, to address unresolved issues concerning the assembly, trafficking, and oligomeric state of the Na pump en route to the plasma membrane. Wild-type and mutant sheep a-subunits will be introduced into the insect cells via the baculovirus system and the pump maturation process will be followed from endoplasmic reticulum to plasma membrane by membrane fractionation and co-immunoprecipitation. In Aim 3, we will confirm and extend our in vitro observations in insect cells to in situ measurements in mammalian cells (i.e. HEK-293 cells). The results from this work will provide important new information about Na,K-ATPase quaternary structure and its importance for exit from the endoplasmic reticulum and delivery to the plasma membrane. We will combine strategies from molecular biology, cell physiology, biochemistry and confocal microscopy to accomplish our scientific goals. The Na,K-ATPase is the pharmacological target for cardiac glycosides, a widely used therapy for congestive heart failure. Considering the mounting evidence that cellular distribution of Na,K-ATPase plays a key role in its physiology, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysregulation of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine and polycystic kidney disease. PUBLIC HEALTH RELEVANCE: The Na,K-ATPase is an essential transport system and the site of action of digitalis, the most widely used therapy to treat patients with congestive heart failure. Prospects for improved therapies for cardiac function, as well as improving impaired renal function, will be greatly aided when we have a better understanding of the regulation of the activity of the Na,K-ATPase in cell membranes and by elucidating the mechanisms by which cells properly deliver this vital enzyme to specific subcellular locations.